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Magdalena Krulova, Katerina Pokorna, Anna Lencova, Jan Fric, Alena Zajicova, Martin Filipec, John V. Forrester, Vladimir Holan; A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient. Invest. Ophthalmol. Vis. Sci. 2008;49(9):3903-3908. doi: https://doi.org/10.1167/iovs.08-1987.
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purpose. To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse.
methods. Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro.
results. Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer.
conclusions. These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12−p63+ population being closer to the primitive limbal SC.
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